North American water shrews, which have traditionally included Sorex alaskanus, S. bendirii, and S. palustris, are widely distributed through Nearctic boreal forests and adapted for life in semiaquatic environments. Molecular mitochondrial signatures for these species have recorded an evolutionary history with variable levels of regional divergence, suggesting a strong role of Quaternary environmental change in speciation processes. We expanded molecular analyses, including more-comprehensive rangewide sampling of specimens representing North American water shrew taxa, except S. alaskanus, and sequencing of 4 independent loci from the nuclear and mitochondrial genomes. We investigated relative divergence of insular populations along the North Pacific Coast, and newly recognized diversity from southwestern montane locations, potentially representing refugial isolates. Congruent independent genealogies, lack of definitive evidence for contemporary gene flow, and high support from coalescent species trees indicated differentiation of 4 major geographic lineages over multiple glacial cycles of the late Quaternary, similar to a growing number of boreal taxa. Limited divergence of insular populations suggested colonization following the last glacial. Characterization of southwestern montane diversity will require further sampling but divergence over multiple loci is indicative of a relictual sky-island fauna. We have reviewed and revised North American water shrew taxonomy including the recognition of 3 species within what was previously known as S. palustris. The possibility of gene flow between most distantly related North American water shrew lineages coupled with unresolved early diversification of this group and other sibling species reflects a complex but potentially productive system for investigating speciation processes.